Reagent kit of global analysis for protein expression and method for qualitative and quantitative proteomic analysis using the same

Abstract

The present invention provides a reagent kit of global analysis for protein expression and method for qualitative and quantitative proteomic analysis using the same, characterized in which isotope labeling reagent is utilized to modify enzymatically cleaved peptides in normal or perturbed cells and subsequently tandem mass spectrometry is used to identify protein sequence, and at the same time, accurately measure the protein expression level based on variation in signal intensity.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention discloses a reagent kit of global analysis for protein expression and method for qualitative and quantitative proteomic analysis using the same, which combines stable isotope labeling and tandem mass spectrometry to accurately quantify protein expression.[0003]2. Description of Related Art[0004]Protein over-expression is often associated with the presence of disease or the administration of drug. Protein regulation is not only related to the transcribed or translated message, but also the post-translational modification. Currently there are three general approaches to the measurement of protein expression in cells: (1) observing the expression of mRNA by microarray to determine whether the post-transcriptional protein is over-expressed. But this method is based on the premises that mRNA expression is positively correlated with protein expression. However in real biological systems, such positive co...

AI Technical Summary

Benefits of technology

"The present invention provides a reagent kit and method for accurate, fast, and economical analysis of protein expression. The reagent kit can label all peptides containing N-terminal amino group or ε-amino group of lysine, and the labeling is done with isotopes of different masses. The reagent kit includes an aldehyde compound and a reducing agent. The aldehyde compound can be modified with isotopes of different masses. The reducing agent is sodium cyanoborohydride. The method involves digesting samples with enzyme, adding the global analysis reagent kit, and analyzing the samples using mass spectrometry. The invention also provides a method for protein de novo sequencing and analysis of phosphoprotein. The technical effects of the invention include accurate and fast analysis of protein expression, identification of protein or quantitative analysis of relative degree of phosphorylation, and analysis of phosphoprotein using immunochromatography or strong cation exchange column to achieve fractionation of protein mixture."

Problems solved by technology

Thus this method cannot cover all types of protein expression and its reproducibility or accuracy is questionable; (2) carrying out quantitative analysis by using two-dimensional electrophoresis and staining gel separated proteins with coomassie blue stain, silver stain, or immunoblotting.

This method is limited by the inherent limitations of the two-dimensional electrophoresis, which shows poor sensitivity and reproducibility in the case of extremely acidic or basic proteins, very large or small proteins, protein expressed at very low level, or membrane proteins; (3) using mass spectrometry in conjunction with chemical reagent (commonly isotope labeling reagent).

But this method concerns the rate of isotope substitution in cells and is unsuitable for in vivo experiment.

But the applications of LC / MS are still limited.

For examples: (1) the large structure of labeling reagent oftentimes poses difficulty to the fragmentation process in tandem mass spectrometry; (2) the high number of atoms of isotope leads to different retention time in chromatographic column for samples containing hydrogen and deuterium, hence resulting in assay error; and (3) ICAT can only tag cysteine-containing peptides, hence cannot be used in global analysis of proteins not containing cysteine.

But the majority of assay reagents being reported in literature have the following drawbacks: (1) the mass spectrometric signals of modified peptides are weakened or prohibited; (2) the incomplete labeling reaction or the exchange between isotopes (hydrogen and deuterium) leads to assay error; and (3) the preparation of assay reagent is tedious and the technique has poor compatibility with other separation techniques (e.g. the different retention time of 1H and 2D labeled peptide fragments in liquid chromatographic column affects the quantitative analysis in mass spectrometry).

But this method is time consuming and not entirely safe.

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